(w/v) exponential gradient SDS-PAGE of the pattern uter membrane proteins after growth of the organism
medium for 2-3 days followed by 3-4 days growth in dlum (lane a); li) normal medium (lane b ) ; ill)
dium alone (lanes c,d).
b e d
M r x 103
92
114
strains examined, including Synechococcus sp. PCC7002, for which siderophore activity has been shown (see Boyer et al., 1987). The general applicability of this method might then be questioned.
Recently, the presence of an hydroxamate siderophore was demonstrated in Synechococcus R2 (D. Laudenbach, personal communication), using an alternative detection system. Further work may be directed towards structural analysis of the siderophore, investigation of the level of extracellular iron which induces siderophore biosynthesis, and Identification of the outer membrane receptor involved in binding the iron-loaded siderophore.
4.3.2 Use of antibodies to investieate the function and export of Synçchocoçcys R2 outer membxans_J>roteing
Western blots of the IRMP showed that these proteins cross-reacted with a polyclonal antisera (see Section 4.2.2) raised against Synechococcus R2 cell walls, isolated from cells grown in normal growth medium (see Fig. 4.3). This antiserum also cross-reacted against a 50,000 molecular weight polypeptide shown to be induced under low sucrose concentrations. Perhaps this polypeptide is multifunctional, or induced under 'general stress' conditions. The presence of antibody to these 'stress response' proteins, suggests that such proteins are present in very small quantities under normal growth conditions, or that they are highly cross- r eactive. Attempts to raise antibodies solely against the approx. 50,000 molecular weight polypeptide, purified by preparative SDS-PAGE, were unsuccessful. The possibility that antibodies to the IRMP might interfere w ith iron uptake and inhibit bacterial growth remains to be
investigated. This would have important implications in bacterial pathogenicity studies.
Western blo t s using a biotin-streptavidin detection system (Section 4.2.3) r e q u i r e d up to S times more primary antibody than those using a system based o n the use of peroxidase conjugated goat anti-rabbit IgG as secondary antibody. It was noteworthy that us ing this latter detection system, h i g h e r levels of non-specific cross-reactivity were observed when washing filters with 0.05% Tween, than us ing Triton X-100.
Recently, a n t i b o d y against CPVI-4 - one of the photosystem II enriched chlorophyll proteins of Synechococcus R 2 , w h ich is particularly abundant in iron-deficient cells (Pakrasi et al., 1985) - was used to identify an iron-regulated membrane protein gene in Synechococcus R2 (Reddy et al., 1987). The antibody against CPVI-4 recognised three proteins of 36,000, 35,000 and 3 4 , 0 0 0 molecular weight in low iron grown membranes. Utilising t h e expression vector Agtll a gene encoding the 36,000 polypeptide w a s identified using antibody as a probe. This protein was shown to be present in the cytoplasmic membrane.
Using a f f i n i t y purified antibody against the 36,000 molecular weight polypeptide ( a kind gift of H. Reithman) we observed cross-reaction with the approx. 35,000 iron-regulated outer membrane protein, together with
some cross-reaction against the 50,000-52,000 outer membrane proteins (see Fig. 4.1 4 ) . Antibody (a kind gift of G. Bullerjahn) against a
carotenoid b i n d i n g protein of the cytoplasmic membrane of Synechocystis sp. PCC6714 (Bullerjahn and Sherman, 1986) also showed some cross reactivity w i t h the 35,000 and 50-52,000 outer membrane proteins of normal and iron-starved Synechococcus R2. In addition, antibody (a gift of H. R e i thman) purified against a 42,000 molecular weight carotenoid- associated thylakoid protein from the cyanobacterium Synechococcus R2
(Masamoto at al., 1987) s h o w e d slight cross-reactivity with Synechococcus R2 outer m e m b r a n e proteins, as well as cross-reactivity with approx. 40,000 and 4 2 , 0 0 0 molecular weight polypeptides o f the
Synechocystls sp. PCC6714 cell wall (see Fig. 4.15). The synthesis of
the 42,000 molecular w e i g h t polypeptide was induced under h igh light intensities, but was a b s e n t or much decreased in iron-stressed cells (Masamoto et al., 1987). The presence o f carotenoids in the
photosynthetic membranes a n d cell walls of Synechococcus R2 is likely to be physiologically i m p ortant in their protection against photo-oxidative damage. It is possible t h a t a family o f such carotenoid-associated proteins exist, which a r e immunochemically related. Immunogold labelling of frozen cell sections using affinity purified antibodies will help clarify the l o c a t i o n and relatedness of these membrane proteins.
The gene encoding the 3 6 , 0 0 0 polypeptide has b een sequenced, but does not appear to possess a signal sequence (K. J. Reddy, personal communication). However, this does not exclude the possibility of the protein being present i n b o t h the cytoplasmic or outer membrane, especially since a signal sequence is not a n absolute requirement for export (see Pugsley a n d Schwartz, 1985).
A Synechococcus R2 m u t a n t lacking this 36,000 molecular weight iron- regulated protein, constr u c t e d by introducing Tn5 into the coding sequence of the gene, s h o w e d that this p rotein was essential for growth in iron-deficient m e d i u m (G. Bullerjahn, personal communication). This
would be consistent w i t h its location in the cell wall, and a function
116
flpurt *.1*
Western blot u s i n g affinitv-purlfled antibody (1: SOOOdilution) against a 36.000 molecular weighs. IftiTfrom S m e c h o c o c c u s R2 . and peroxidase coniu& a t e d . .ggas anti-rabbit IeG (1:10.000 dilution) as secondary
antibody
Lanes a , b - Synechococcus R2 OMP, chelator-deficient medium; lane c -
118
Figure 4.15 Western blot using antibody (1:1000 dilution') purified against a 4 2 .000 molecular w e i g h t carotenoid-associated thvlakoid protein from 5ynechocgccug_R2
Peroxidase conjugated goat anti-rabbit IgG (1:10,000 dilution) was used as secondary antibody. Lane a - Synechococcus R2 OMP, chelator-
deficient mediun; lane b - Synechococcus R2 OMP, normal medium; lanes c,d - Synechococcus sp. PCC7002 OMP, normal medium; lane e - Synechocyscis sp. PCC6714 OMP, normal medium.
a
b
c
d
e
«
M r x 10°
Antibodies raised against Synechococcus R2 cell walls were u s e d to
analyse the export of the outer membrane proteins. Soluble, thylakoid membrane, cytoplasmic membrane and cell wall protein fractions of
Synechococcus R2 were separated by SDS-PAGE, transferred to nitrocellulose, arid the Western blots screened using ant i s e r a to
Synechococcus R2 cell walls. Cross reaction was specific t o the cell
wall fraction of Synechococcus R2, and no reaction against a n y cytoplasmically located unprocessed form of the outer m e m b r a n e proteins was observed (see Fig. 4.16). This may be a result of the instability of the outer membrane proteins in the cytoplasm, or that t h e y are not immunogenic in the unprocessed form (assuming that these pr o t e i n s possess a leader sequence). Alternatively, the quantity o f outer membrane protein precursor in the cytoplasm may be undetectable.
4.3.3 Screening 9f a gyngchococgug. R2 A g t U library
Using a genomic library of Synechococcus R2 in Agtll (a k i n d gift of K. J. Reddy) 20-30 positive plaques were identified using the polyclonal antisera to Synechococcus R2 cell walls (which h a d b een preabsorbed against a bound bacterial lysate - see Section 4.2.4), a f ter
125
autoradiography using I-labelled secondary antibody (see Fig. 4.17). This m e t h o d produces fusion proteins synthesised in the A g t l l
recombinant phage which can be recognised by antibody screening. These positive plaques await further analysis, but it should be possible to clone genes encoding the major outer membrane proteins of Synechococcus
120
figvrg ¿.IS Use .of SynecAococcus R2 cell wall antibody to study the